The present invention relates generally to a technique for analyzing biochemical substances, and especially to an absorptiometric method for measuring existance, concentrations and/or activities of chemical substances in sample liquids in an accurate manner by measuring variations in absorbance with respect to time.
Such a method of measuring the concentrations or activities of substances contained in a sample by detecting a variation in absorbance or optical density of a test liquid which is a mixture of the sample and one or moe reagents is sometime called a kinetic assay and has been used for measuring enzymes such as Glumatate Oxalacetate Transaminase (GOT), Glumatate Pyruvate Transaminase (GPT), and the like. In a known apparatus for effecting such a kinetic assay, only one photometering position is arranged and the variation in absorbance is measured successively or intermittently while a cuvette containing the test liquid is kept stably at the photometering position for a given time period. Then concentrations and/or activities of chemical substances in sample liquid are calculated according to the measured variations of absorbance. However, in order to perform a highly precise analysis, it is necessary or advantageous to measure the large variation of absorbance. For this purpose it is necessary to keep respective samples at the photometering position for a sufficiently long time. Therefore, such analyzing apparatus has a slow feeding speed and thus, there is a drawback that a sample processing or treating ability is low.
In order to overcome such a drawback, there has been proposed an analyzing apparatus comprising a plurality of photometering positions aranged along a reaction line. While cuvettes containing test liquids which are mixtures of samples and reagents are fed along the reaction line continuously or intermittently, the absorbances of the test liquids are measured only once at respective photometering positions. Then, the variation in absorbance can be derived as a difference between absorbance values measured at any two photometering positions. In such an analyzing apparatus, since the sample absorbance is photometered at a plurality of photometering positions, it is not necessary to remain the cuvettes at the respective photometering positions for a long time and thus, the processing speed can be made high. However, in such a method, a plurality of measuring channels each belonging to respective photometering positions have different characteristics due to difference in various factors such as stray light, electrical characteristics of pre-amplifier, and deviation in light axis with respect to a cuvette position. These differences result in variation of linearity of calibration curve of respective photometering channels. Moreover, in the kinetic assay, since variations of absorbances of reagents with respect to time cause an error in analyzed results, it is necessary to compensate the absorbance variations of the reagents with respect to time.
Therefore, in such an analyzing apparatus, an analyzing operation has been usually performed according to the following steps;
(1) Adjustment of Apparatus at 0% and 100%. PA1 (2) Measurement of Reagent Blank; the absorbance variation per unit time .DELTA.E.sub.B of the reagent blank liquid (reagent per se) is measured.
(3) Calculation of Concentration or Activity Converting Coefficient I; in one case the coefficient is calculated from an equation I=V.sub.t /(.epsilon..times.d.times.v.sub.s), wherein v.sub.s, v.sub.r, d, and .epsilon. are sample amount, reagent amount, optical path length, and atomic absorption coefficient, respectively, and V.sub.t indicates v.sub.s +v.sub.r. In another case the absorbance variation per unit time .DELTA.E.sub.s is obtained by using a standard liquid or a controlling serum with known concentration (activity) c as the sample liquid and then the coefficient is calculated from an equation I=c/(.DELTA.E.sub.s .DELTA.E.sub.B).
(4) Measurement of Unknown Sample; the absorbance variation per unit time .DELTA.E.sub.v is measured and then a measuring result i.e. a concentration c.sub.x of a substance to be measured is calculated from a formula c.sub.x =I.times.(.DELTA.E.sub.v .DELTA.E.sub.b).
The reagent blank mentioned above is generally obtained by performing the photometry upon the reagent itself by a plurality of times on the basis of a light flux passing through air or water. In the analyzing apparatus having a plurality of measuring channels, at first, the reagent absorbance is measured at respective photometering positions by delivering only reagent into the cuvette on the basis of a transmittivity of air or water and then the reagent blank is calculated from a difference between the reagent absorbances measured at the different photometering position. In this case, if the linearity of calibration curves at respective photometering positions is not strictly identical with each other, it is not possible to obtain accurately the reagent blank from the difference between the reagent absorbances measured at different photometering positions. In order to overcome such a drawback, if the analysis is performed by measuring the absorbance variation with respect to time for all samples including the reagent blank at two fixedly predetermined photometering positions, it is possible to use the reagent blank as a difference in absorbance variation between these photometering positions, because in this case the difference in analyzing property between the phomometering positions maybe considered to be always constant. Then the reagent blank is stored in a memory and the absorbance differences of samples can be corrected by the stored reagent blank. However, in case of performing the analysis by selecting suitable absorbances measured at any two photometering positions, since the two photometering positions may be different for respective samples, it is necessary to store a very large number of absorbance variations as the reagent blanks. For instance, when an amount of substances to be measured is small, it is preferable to derive a difference in absorbance values measured at two photometering positions which are spaced farthest, but when an amount of substance is abnormally large, it is advantageous to calculate difference in absorbance values measured at two adjacent photometering positions. Further, even in case of using the absorbance values between adjacent photometering positions, measurement timings in the reaction procedure should be made different for respective samples. In order to satisfy such requirements, it is necessary to store a very large number of reagent blanks calculated from all differenes in absorbance values between all the photometering positions. For instance, when there are provided fifteen photometering positions along the reaction line, more than hundred reagent blanks have to be stored in the memory. Therefore, the memory is liable to be large and a control circuit for the memory becomes very complicated.